Sildenafil increases EE cell density in colon mucosa of healthy mice but has no effect on transit time or fecal water content.

<p>(A) Quantification of enteroendocrine cells (CgA) from stained tissue sections from colons of mice treated for five days with sildenafil, linaclotide, or untreated (Ctrl). (B) Quantification of enteroendocrine cells in colons from mice treated with various doses of sildenafil <i>ad libitum</i> in drinking water for five days. (C) CgA-positive cells over time following <i>ad libitum</i> sildenafil treatment. C57/BL6 mice were treated with sildenafil or linaclotide for five days prior to intestinal transit assay of (D) total transit or (E) upper intestinal transit. (F) Fecal pellets were collected from transit assays and measured for fecal water content. Data are shown as means, error bars represent SEM, n = 3 (A-C), n = 12 (D-E). *P<0.05, **P<0.005 by two-tailed Student’s t-test, each treatment group is compared to control (A, B) or time 0 (C).</p

Sildenafil normalizes transit in loperamide-induced constipation model.

<p>Mice were either provided vehicle (healthy) or 10 mg/kg loperamide (constipated). (A) Total intestinal transit, (B) distance traveled by a gavaged bolus of charcoal, and (C) fecal water content were measured in untreated (Ctrl) or sildenafil treated (Sild) mice as indicated. Data are shown as means, error bars represent SEM, n = 12 (A-C). *P<0.05, ***P<0.001 by one-way ANOVA and Tukey’s post hoc analysis.</p

Sildenafil decreases intestinal transit time in a DSS-recovery model of IBS.

<p>(A,B) Quantification of 5-HT-positive cells in stained sections of colon from healthy mice, or those recovered from DSS treatment (DSS recovery). Mice were either untreated controls (Ctrl) or treated with sildenafil for five days (Sild). (C) Intestinal transit time and (D) distance traveled by a gavaged bolus of charcoal were measured in healthy and DSS-recovery mice. Mice were either untreated (Ctrl), or treated with sildenafil or linaclotide acutely (1 hr; Acute Sild, Acute Lin respectively) and 5-days or (Sild, Lin respectively). Data are shown as means, error bars represent SEM, n = 6 (B), n = 12 (C-D). *P<0.05, ** P<0.005, ***P<0.001 by one-way ANOVA and Tukey’s post hoc analysis.</p

Sildenafil regulates fecal water content in DSS-recovery mice.

<p>Fecal pellets from healthy mice, or those recovered from DSS treatment (DSS recovery) were collected to measure (A) total wet and dry mass of pellets and (B) fecal water content. Mice were either untreated (Ctrl), or treated with sildenafil or linaclotide acutely (1 hr; Acute Sild, Acute Lin respectively) or 5-days (Sild, Lin respectively). (C) Mice were either provided water (healthy) or 3% DSS for 5 days (DSS). Barrier function was assessed using the FITC-dextran approach as described in the Materials and Methods. Mice exposed to DSS were either untreated (Ctrl) or treated with an acute dose of sildenafil (Sild). (D) Levels of tight junctional proteins claudin 4 and occludin were measured by western blot in mucosa collected from untreated mice (Ctrl) and mice treated sildenafil for 5 days (Sild). Data are shown as means, error bars represent SEM, n = 12 (A, B), n = 6 (C, D). *P<0.05, ***P<0.001 by one-way ANOVA and Tukey’s post hoc analysis.</p

PDE-5 inhibitors increase cGMP and regulate homeostasis in the colon epithelium.

<p>(A) cGMP levels in the intestinal mucosa of untreated mice (Ctrl), or 5 h following gavage (Gav) or intraperitoneal injection (IP) of vardenafil or sildenafil, or mice provided sildenafil in water <i>ad libitum</i> (Sild Water) for 5 days. (B) Daily intake by mice provided water alone (Control) or water containing 35μg/ml sildenafil. (C, D) Quantification of goblet cells (ABPAS) and (E, F) Ki-67- positive cells in colon mucosa of CD-1 mice treated with vardenafil IP (vard) or sildenafil in drinking water (sild) for 5 days. Data are shown as means, error bars represent SEM, n = 6 (A, B), n = 3 (D, F). *P<0.05, ***P<0.001 by two-tailed Student’s t-test.</p